Bauhinia extract and uses thereof

ABSTRACT

Disclosed herein is the novel use of use of a  Bauhinia  spp. extract, which may upregulate neprilysin, induce autophagy, protect neuron from amyloidopathy or tauopathy, and/or promote neurite outgrowth, thus the  Bauhinia  spp. extract of the present disclosure may be used as a dietary supplement for the prophylaxis or treatment of amyloid related neurodegenerative diseases so as to ameliorate or alleviate symptoms associated with the amyloid related neurodegenerative diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/772,810, filed May 1, 2018, which is an application U.S. NationalStage Filing under 35 U.S.C. 371 from International Patent ApplicationSerial No. PCT/US2016/060359, filed Nov. 3, 2016, and published on May11, 2017, which claims the priority of U.S. Ser. No. 62/251,638, filedNov. 5, 2015, the disclosure of which are incorporated by referenceherein in their entireties.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure relates in general, to the novel use of aBauhinia spp. extract. More specifically, the present disclosure relatesto the use of Bauhinia spp. extract as a dietary supplement for thetreatment of a neurodegenerative disease.

2. Description of Related Art

Bauhinia variegata Linn. (Fabaceae) is traditionally prescribed inindigenous medicines in India. Various parts of the plant (flower buds,flowers, stem, stem bark, leaves, seeds, and roots) are used in thetreatment of asthma, jaundice, tuberculosis, leprosy, and skin diseases.It has also been reported that the stem bark of B. variegata possessantitumor, anti-ulcer, and antibacterial activities. Recent studies inanimal models further demonstrated that the ethanol extract of B.variegata (BVEE) exhibited significant nephroprotective effect againstcisplatin-induced nephropathy, and antidiabetic activity inalloxan-induced hyperglycemic activities.

Alzheimer's disease (AD) is a progressive neurodegenerative disease andis the most prevalent form of age-related dementia. It is widelybelieved that the accumulation of Aβ and Tau in AD is the causativeevent triggering neurodegeneration and related pathogenic pathways,which together contribute to the onset and progression of AD. Thetruncation of Tau protein has been identified in human sporadic AD, andcould also instigate the aggregation of Tau and formation ofneurofibrillary tangles in AD. Accumulated evidence indicated thatneprilysin (NEP) is the primary endopeptidase responsible for theclearance of Aβ and that its functional deficiency is evident duringaging process and AD. Consistent with this notion, an inversecorrelation is observed between NEP levels and brain Aβ loads in NEPknock-out mice (20-24). NEP has thus been regarded as the most promisingpharmacological target for the development of therapeutics aiming topromote Aβ degradation as AD treatments (25-30). Furthermore, autophagicvacuoles has been shown to be the major intracellular compartment forTau clearance. This autophagy-mediated degradation system is alsobelieved to play a significant role in controlling the homeostasis ofAβ. Consistent with its protective role in neurons, defective autophagyhas been implicated in the pathogenesis of various neurodegenerativediseases, including AD. Emerging findings suggest that autophagosomescan engulf damaged organelles and aberrant protein aggregates for theclearance of these internalized cargos following fusion with lysosomes.It is thus reasonable to speculate that autophagy-inducing agents couldrender neuroprotective efficacy through promoting the autophagy-mediatedclearance of aberrant protein aggregates, including Aβ and Tau.

By use of a zebrafish model of tauopathy, a cell-based assay forneprilysin, and a cell-based assay for autophagy, inventors of thepresent disclosure unexpectedly discovered that Bauhinia spp. extractcan simultaneously inhibit tauopathy-elicited neurotoxicity, enhanceneprilysin activity, and induce autophagic activity, thus the presentidentified Bauhinia spp. extract could be used as a health food dietarysupplements to improve overall memory and/or cognitive function of apatient suffering from a neurodegenerative disease.

SUMMARY

The present disclosure relates to novel use of a Bauhinia spp. extractin upregulating neprilysin, inducing autophagy, protecting neuron fromamyloidopathy or tauopathy, and/or promoting neurite outgrowth, thus theBauhinia spp. extract of the present disclosure may be used as a dietarysupplement for the prophylaxis or treatment of amyloid relatedneurodegenerative diseases so as to ameliorate or alleviate symptomsassociated with the amyloid related neurodegenerative diseases.

Accordingly, it is the first aspect of the present disclosure to providea method for the treatment of a subject having or suspected of having anamyloid related neurodegenerative disease. The method includes the stepof, orally administering to the subject a therapeutically effectiveamount of a Bauhinia spp. extract to ameliorate or alleviate symptomsassociated with the amyloid related neurodegenerative diseases.

According to embodiments of the present disclosure, the Bauhinia spp.may be Bauhinia variegate or Bauhinia×blakeana.

According to embodiments of the present disclosure, the Bauhinia spp.extract may be a water extract or an ethanol extract.

According to some embodiments of the present disclosure, the waterextract of Bauhinia spp. is produced by mixing the powdered stem ofBauhinia spp. with water at a ratio of 1:5 (w/v) to 1:20 (w/v) at about30-100° C. for about 0.5-5 hrs. Preferably, the water extract ofBauhinia spp. is produced by mixing the powdered stem of Bauhinia spp.with water at a ratio of 1:10 (w/v) at about 50° C. for about 2 hrs.

According to further embodiments of the present disclosure, the ethanolextract of Bauhinia spp. is produced by mixing the powdered stem ofBauhinia spp. with 95% ethanol (vol %) at a ratio of 1:5 (w/v) to 1:20(w/v) at about 15-35° C. for about 1-10 days. Preferably, the ethanolextract of Bauhinia spp. is produced by mixing the powdered stem ofBauhinia spp. with 95% ethanol (vol %) at a ratio of 1:10 (w/v) at about25° C. for about 7 days.

According to embodiments of the present disclosure, the Bauhinia spp.extract is orally administered to the subject in the amount of about5-50 mg/Kg. Preferably, the Bauhinia spp. extract is orally administeredto the subject in the amount of about 20 mg/Kg.

According to embodiments of the present disclosure, theneurodegenerative disease that may be treated by the present Bauhiniaspp. extract is selected from the group consisting of, Alzheimer'sdisease (AD), Huntington's disease (HD), vascular dementia,frontotemporal dementia, semantic dementia and dementia with Lewybodies, amyolateral sclerosis (ALS), and Parkinson's disease (PD).

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Other features and advantages of theinvention will be apparent from the detail descriptions, and fromclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings, where:

FIG. 1. Treatment of Bauhinia extract could decrease neuronal death byoverexpression of human tau-GFP in zebrafish embryo. A. The expressionconstruct pHuC-hTau-GFP was first injected into zebrafish embryos at1-cell stage. Then, GFP-labeled neuronal cells were observed at 24, 48and 72 hpf. Some GFP signals were observed in neuronal cells of 24 hpfembryos, then broken down as small dots and disminished in 48 hpfembryos. However, there were other GFP signals remained intact inneuronal cells. B. Neuronal numbers with GFP signals in embryos at 48hpf were counted and separated into two groups, lower than 2 neurons(0-2) and more neurons between 3-5. Treatment of BVEE enhanced embryoswith more neurons (FIG. 1, panel a) with 46% (FIG. 1, panel B);

FIG. 2. Treatment with BVEE induces the promotion of neurite outgrowthin zebrafIsh embryo. A. The expression plasmid pHuC-GFP was firstinjected into zebrafish embryos at one-cell stage. Eight hours afterinjection, those injected embryos were treated with BVEE or DMSO ascontrol. B. zebrafish numbers with significant neurite outgrowth werecounted 40 hrs after treatment;

FIG. 3. Treatments with BVEE induces autophagy. A. GFP-LC3-HEK cellswere treated with BVEE (50 μg/ml) at 37° C. for 24 h. An autophagyinducer, rapamycin, and an autophagy inhibitor, 3-MA, were included ascontrols. Clarified lysates containing equal amounts of proteins wereresolved by SDS-PAGE and analyzed by Western blotting with anti-p62 (toppanel), anti-LC3 (middle panel), and anti-GAPDH (bottom panel)antibodies. B. A stable cell line p62-RL-HEK that overexpresses Renillaluciferase-tagged p62 was treated with BVEE (50 μg/ml) at 37° C. for 24h. Additional treatments with either rapamycin or 3-MA were included ascontrols. Renilla luciferase signal was determined by the addition ofcoelenterazine at equal volume to a final concentration of 5 μM.Luminescence emitted by DMSO-treated cells (Control) was referred to as100% relative Renilla luciferase signal;

FIG. 4. Effect of BVEE on NEP activity. The line graph illustrates thedose dependency of BVEE on NEP activity in accordance with oneembodiment of the present disclosure;

FIG. 5. The viability of p62-RL-HEK cells in response to BVEE treatment.The p62-RL-HEK cells were treated with or without BVEE (50 or 100 μg/ml)at 37° C. for 24 h. Additional treatments with either rapamycin or 3-MAwere included as references. Viable cells was determined by the additionof CellTiter 96 AQueous non-radioactive cell proliferation assayreagents as described in Methods. Absorbance at 490 nm by DMSO-treatedcells (Control) was referred to as 100% relative viability.

FIG. 6. Aβ42-injected AD mice receiving BVEE exhibit cognitiveimprovement. AD mice treated with BVEE can remember the location ofhidden platform comparable to the performance of control mice (escapelatency) in Morris water maze test. Non-operation mice (Noinjection/solvent), n=5; Sham-operated mice treated with solvent (PBSinjection/solvent), n=5; Aβ42-injected mice treated with solvent (Aβ42injection/solvent), n=8; Aβ42-injected mice treated with BVEE (Aβ42injection/BVEE), n=6.

FIG. 7. BVEE induces activation of autophagy in hippocampus ofAβ42-injected mice. Male C57BL/6 mice were intracerebrally injected withAβ42 and treated with vehicle alone (0.1% DMSO) or BVEE (250 mg/Kg/d)for 2 months. After the cognitive function of mice were analyzed byMorris water maze test, brains of mice were then processed forfluorescence immunohistochemistry analysis with anti-p62. DAPI stainingwas performed to visualize nucleus. Representative images offluorescence immunohistochemical analysis show the levels of p62 (red)and DAPI staining (blue) in the hippocampal region of BVEE-treated anduntreated mice (vehicle). Quantitative data are shown as average p62levels (±SD) from 3 consecutive sections (15 μm thick) of mouse brain(n=3).

FIG. 8. Long-term BVEE treatment improved the cognitive status of theAPP/PSI transgenic mice. 3-month-old APP/PS1 mice were orally treatedwith 250 mg/Kg·BW BVEE or saline 6 times a week for 7 months. Spatialand reference memories of the saline-treated APP/PS1 mice (n=12),BVEE-treated APP/PS1 mice (n=8) and WT littermates (n=15) were thenassessed by (A) spatial acquisition trials and (B) probe trial of Morriswater maze test at 10 months of age. Data was expressed in mean±SEM.Significance between different treatment groups calculated by two-wayANOVA with Fisher's LSD test are shown at the right side of the curves.Data of the WT group and the BVEE-treated group on each day werecompared with the saline-treated APP/PS1 mice. * P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001, ns, not significant.

FIG. 9. Amyloid plaque burdens of APP/PSI mice were decreased after BVEEtreatment. Plaque burdens of the 11-months-old WT littermate,saline-treated and BVEE-treated APP/PS1 mice were assessed by (A) ELISAand (B) thioflavin S staining. (A) After euthanasia, cerebral cortex andhippocampus were isolated for ELISA. Insoluble human Aβ40 and Aβ42levels were quantified by commercial ELISA kit (n=6 for BVEE treatment;n=5 for saline treatment; n=3 for WT mice). (B) Plaque burdens werefurther visualized by thioflavin S staining.

Scale bar represents 800 μm. (C) Plaque number, total plaque area, andpercentage covered by plaques per ThS-stained section were quantitatedby ImageJ. Data was expressed in mean±SEM. Significant andnot-significant (ns) results compared with the saline-treated APP/PS1mice were demonstrated. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001were calculated by one-way ANOVA with Fisher's LSD test (n=12 sectionsfrom 2 mice with BVEE treatment; n=40 sections from 7 mice with salinetreatment; n=10 sections from 5 WT mice).

FIG. 10. Liver and kidney toxicity test for the long-term BVEEtreatment. Blood samples of the BVEE-treated APP/PS1 mice werecollected, and AST, ALT, ALP, BUN, CRE levels were then analyzed. Allresults were expressed in mean±SEM (n=3 for BVEE treatment; n=4 forsaline treatment). * P<0.05, compared with saline-treated APP/PS1 micewere calculated by ANOVA with Fisher's LSD test. Not significant; ns.The range of AST, ALT, ALP, BUN, CRE levels obtained from 10-wk-old ICRmice (n=10) are shown in dash line.

FIG. 11. NEP activity assay. This bar graph illustrates the respectiveeffects of extracts of Bauhinia×blakeana Dunn. (BB) prepared by 95%ethanol, 40% ethanol, and water on NEP activity in accordance with oneembodiment of the present disclosure;

FIG. 12. The autophagy-inducing potency of extracts derived from B.variegate (BV) by using different solvents. The p62-RL-HEK cells weretreated with or without B. variegate extract (50 mg/ml) at 37° C. for 24h. Additional treatments with either rapamycin (RAP) or 3-MA wereincluded as references. Renilla luciferase (Luc) activity was determinedby the addition of coelenterazine to a final concentration of 5 mM.Luminescence emitted by DMSO-treated cells (Control) was referred to as100% relative Renilla luciferase signal. Extracts derived from differentsolvents were lyophilized, and redissolved in DMSO for subsequentanalysis. Capital letter denotes the solvent used in the extraction ofB. variegate. A, hexane; B, ether; C, ethyl acetate; D, hot water; E,ethanol.

DESCRIPTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example may beconstructed or utilized. The description sets forth the functions of theexample and the sequence of steps for constructing and operating theexample. However, the same or equivalent functions and sequences may beaccomplished by different examples.

1. Definitions

For convenience, certain terms employed in the context of the presentdisclosure are collected here. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of the ordinary skill in the art to which thisinvention belongs.

Unless otherwise defined herein, scientific and technical terminologiesemployed in the present disclosure shall have the meanings that arecommonly understood and used by one of ordinary skill in the art. Unlessotherwise required by context, it will be understood that singular termsshall include plural forms of the same and plural terms shall includethe singular. Specifically, as used herein and in the claims, thesingular forms “a” and “an” include the plural reference unless thecontext clearly indicates otherwise. Also, as used herein and in theclaims, the terms “at least one” and “one or more” have the same meaningand include one, two, three, or more.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in therespective testing measurements. Also, as used herein, the term “about”generally means within 10%, 5%, 1%, or 0.5% of a given value or range.Alternatively, the term “about” means within an acceptable standarderror of the mean when considered by one of ordinary skill in the art.Other than in the operating/working examples, or unless otherwiseexpressly specified, all of the numerical ranges, amounts, values andpercentages such as those for quantities of materials, durations oftimes, temperatures, operating conditions, ratios of amounts, and thelikes thereof disclosed herein should be understood as modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the present disclosureand attached claims are approximations that can vary as desired. At thevery least, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques. Ranges can be expressed herein as from oneendpoint to another endpoint or between two endpoints. All rangesdisclosed herein are inclusive of the endpoints, unless specifiedotherwise.

The term “treatment” as used herein includes preventative (e.g.,prophylactic), curative or palliative treatment; and “treating” as usedherein also includes preventative (e.g., prophylactic), curative orpalliative treatment. In particular, the term “treating” as used hereinrefers to the application or administration of the present molecularconstruct or a pharmaceutical composition comprising the same to asubject, who has a medical condition a symptom associated with themedical condition, a disease or disorder secondary to the medicalcondition, or a predisposition toward the medical condition, with thepurpose to partially or completely alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of said particulardisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder and/orcondition, and/or to a subject who exhibits only early signs of adisease, disorder and/or condition, for the purpose of decreasing therisk of developing pathology associated with the disease, disorderand/or condition.

The term “therapeutically effective amount” as used herein refers to thequantity of the present recombinant protein that is sufficient to yielda desired therapeutic response. A therapeutically effective amount of anagent is not required to cure a disease or condition but will provide atreatment for a disease or condition such that the onset of the diseaseor condition is delayed, hindered or prevented, or the disease orcondition symptoms are ameliorated. The therapeutically effective amountmay be divided into one, two or more doses in a suitable form to beadministered at one, two or more times throughout a designated timeperiod. The specific effective or sufficient amount will vary with suchfactors as the particular condition being treated, the physicalcondition of the patient (e.g., the patient's body mass, age, orgender), the type of mammal or animal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compounds or itsderivatives. The therapeutically effective amount may be expressed, forexample, as the total mass of the drug, i.e., the Bauhinia spp. extractin grams, milligrams or micrograms; or a ratio of mass of the drug tobody mass, i.e., the Bauhinia spp. extract as milligrams per kilogram(mg/kg). Specifically, the term “therapeutically effective amount” usedin connection with the Bauhinia spp. extract described herein refers tothe quantity of the Bauhinia spp. extract, which is sufficient toupregulate neprilysin, induce autophagy, protect neuron fromamyloidopathy or tauopathy, and/or promote neurite outgrowth, therebyalleviate or ameliorate the symptoms associated with theneurodegenerative disease in the subject. Persons having ordinary skillscould calculate the human equivalent dose (HED) for the medicament (suchas the compounds of the present disclosure) based on the dosesdetermined from animal models set forth in the working examples of thepresent disclosure. For example, one may follow the guidance forindustry published by US Food and Drug Administration (FDA) entitled“Estimating the Maximum Safe Starting Dose in Initial Clinical Trialsfor Therapeutics in Adult Healthy Volunteers” in estimating a maximumsafe dosage for use in human subjects.

The terms “administering or administration” as used herein refers to theapplication of a Bauhinia spp. extract or a pharmaceutical compositioncomprising the same to a subject in need of a treatment thereof.

The terms “subject” and “patient” are used interchangeably herein andare intended to mean an animal including the human species that istreatable by the molecular construct, pharmaceutical composition and/ormethod of the present invention. The term “subject” or “patient”intended to refer to both the male and female gender unless one genderis specifically indicated. Accordingly, the term “subject” or “patient”comprises any mammal, which may benefit from the treatment method of thepresent disclosure. Examples of a “subject” or “patient” include, butare not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat,cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, thepatient is a human. The term “mammal” refers to all members of the classMammalia, including humans, primates, domestic and farm animals, such asrabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals;and rodents, such as mouse and rat. The term “non-human mammal” refersto all members of the class Mammalis except human.

2. Preparation of Bauhinia Spp. Extract

The present invention is based on the discovery that the Bauhinia spp.extract prepared in according to procedures described in the workingexample of the present disclosure is capable of upregulating neprilysin,inducing autophagy, protecting neuron from amyloidopathy or tauopathy,and/or promoting neurite outgrowth, and exerts no toxic effects towardmetabolite enzymes and/or renal functions, thereby may be used as adietary supplement to alleviate or ameliorate the symptoms associatedwith a neurodegenerative disease in a subject in need of such treatment.

Accordingly, the first aspect of the present disclosure is to provide aBauhinia spp. extract, which is useful for the treatment of a subjecthaving or suspected of having a neurodegenerative disease.

Examples of suitable species of Bauhinia spp. for use in the presentdisclosure include, but are not limited to, Bauhinia variegate andBauhinia×blakeana Dunn.

In one example, the Bauhinia spp. extract of the present disclosure isprepared from the fresh or dried powdered stem of Bauhinia variegate. Inanother example, the Bauhinia spp. extract of the present disclosure isprepared from the fresh or dried powdered stem of Bauhinia×blakeanaDunn. According to embodiments of the present disclosure, the driedpowdered stem of Bauhinia spp. is mixed with a non-toxic solvent in aratio of about 1:5 (w/v) to 1:20 (w/v) at suitable temperature forvarious periods of time so as to produce an extract that contains activecomponents, i.e., components that upregulate neprilysin (NEP) activity,or promote the neurite outgrowth.

According to some embodiments of the present disclosure, the presentBauhinia spp. extract is produced by mixing the powdered stem ofBauhinia spp. with water at a ratio of 1:5 (w/v) to 1:20 (w/v), such as1:5, 1:6 1:7, 1:8, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, and 1:20 (w/v), at a temperature of about 30-100° C., such as 30,40, 50, 60, 70, 80, 90, and 100° C. for a period of about 0.5-5 hrs,such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, and 5 hrs. Preferably, theBauhinia spp. is produced by mixing the powdered stem of Bauhinia spp.with water at a ratio of 1:8 (w/v) to 1:15 (w/v), such as 1:8, 1:10,1:11, 1:12, 1:13, 1:14, and 1:15, (w/v), at a temperature of about40-80° C., such as 40, 50, 60, 70, and 80° C. for a period of about 1-3hrs, such as 1, 2, and 3 hrs. Most preferably, the Bauhinia spp. isproduced by mixing the powdered stem of Bauhinia spp. with water at aratio of 1:10 (w/v) at about 50° C. for about 2 hrs.

According to other embodiments of the present disclosure, the presentBauhinia spp. extract is produced by mixing the powdered stem ofBauhinia spp. with 95% ethanol (vol %) at a ratio of 1:5 (w/v) to 1:20(w/v), such as 1:5, 1:6 1:7, 1:8, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, and 1:20 (w/v), at a temperature of about 15-35°C., such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, and 35° C. for about 1-10 days. Preferably, thepresent Bauhinia spp. extract is produced by mixing the powdered stem ofBauhinia spp. with 95% ethanol (vol %) at a ratio of 1:8 (w/v) to 1:15(w/v), such as 1:8, 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15 (w/v), at atemperature of about 20-30° C., such as 20, 21, 22, 23, 24, 25, 26, 27,28, 29, and 30° C. for about 3-8 days. Most preferably, the presentBauhinia spp. extract is produced by mixing the powdered stem ofBauhinia spp. with 95% ethanol (vol %) at a ratio of 1:10 (w/v) at about25° C. for about 7 days.

The Bauhinia spp. extract thus obtained is preferably freeze dried orlyophilized and stores in a cool, dry and light-proof environment untiluse.

3. Use of the Bauhinia Spp. Extract

According to embodiments of the present disclosure, the Bauhinia spp.extract prepared in accordance with the method of the present disclosureis capable of reducing tauopathy induced neuron toxicity (see Example1), promoting neurite outgrowth (see Example 2), activating autophagy(see Example 3), and upregulating neprilysin (NEP) activity (see Example4) in vitro; improving memory and cognitive functions (see Examples 6and 7) in vivo. Most importantly, it does not impair metabolic enzymeactivity or renal function (see Example 8), nor does it exert anycytotoxicity (see Example 5). Thus, the present Bauhinia spp. extractmay be sued as a dietary supplement to treat a subject having aneurodegenerative disease, particularly an amyloid relatedneurodegenerative diseases, to improve the overall memory and cognitivefunctions of the subject.

Accordingly, it is the second aspect of the present disclosure toprovide a method of treating a subject having or suspected of having aneurodegenerative disease. The method includes the step of, orallyadministering to the subject a therapeutically effective amount of thepresent Bauhinia spp. extract to ameliorate or alleviate symptomsassociated with the amyloid related neurodegenerative diseases.

Examples of neurodegenerative disease that may be treated by the presentBauhinia spp. extract include but are not limited to, Alzheimer'sdisease (AD), Huntington's disease (HD), vascular dementia,frontotemporal dementia, semantic dementia and dementia with Lewybodies, amyolateral sclerosis (ALS), and Parkinson's disease (PD).

The Bauhinia spp. extract useful with the present method may be a waterextract or an ethanol (i.e., 95% ethanol) extract of Bauhinia variegateor Bauhinia×lakeana. In some examples, the 95% ethanol extract ofBauhinia variegate is employed; whereas in other examples, the waterextract of Bauhinia variegate or Bauhinia×blakeana is preferred.

According to embodiments of the present disclosure, the Bauhinia spp.extract is orally administered to the subject in a single dose or inmultiple doses (e.g., 2, 3 or 4 doses) at an amount of about 5-50mg/Kg/day, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 mg/Kg/day.Preferably, the Bauhinia spp. extract is orally administered to thesubject in the amount of about 10-40 mg/Kg/day, such as 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39 and 40 mg/Kg/day. More preferably, theBauhinia spp. extract is orally administered to the subject in theamount of about 15-35 mg/Kg/day, such as 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 mg/Kg/day. Mostpreferably, the Bauhinia spp. extract is orally administered to thesubject in the amount of about 20 mg/Kg/day.

The present Bauhinia spp. extract may be manufactured into dosageformulations suitable for oral administration, such as powders, tablets,caplets, capsules, solutions, and/or suspensions, by mixing suitableamounts of the lyophilized Bauhinia spp. extract with pharmaceuticallyacceptable excipients, binders, disintegrants, dispersants, diluents,lubricants, flavoring agents, and/or coloring agents to produce thedesired oral formulations. For example, the dried granules of thepresent Bauhinia spp. extract may be directly compressed intopharmaceutically flash-melt oral dosage forms, e.g., tablets, caplets,and the like by mixing with suitable excipients. Alternatively, thedried granules of the present Bauhinia spp. extract may be encapsulatedwithin gelatin capsules. Still optionally, the present Bauhinia spp.extract may be dissolved or suspended in a suitable solvent, such aswater, a buffer solution or elixir to produce orally administered liquidor suspension.

The present invention will now be described more specifically withreference to the following embodiments, which are provided for thepurpose of demonstration rather than limitation.

Examples

Materials and Methods

Preparation of Crude Bauhinia Extract

The air-dried, 100 g powdered stems of B. variegate (BV) orBauhinia×blakeana Dunn. (BB) were extracted at a ratio of 1:10 (w/v)with 95% ethanol (BVEE or BBEE) on a rotary shaker (150 rpm) for 7 daysat room temperature. Water extraction was carried out in H₂O (1:10; w/v)for two hours at 50° C.

Extracts of B. variegate (BV) using organic solvents such as hexane,ether, and ethyl acetate (EA) were also prepared in similar fashionaccording to ethanol based extraction procedures. All the filtrates werelyophilized and stored at −20° C.

Cell Culture

Human embryonic kidney (HEK) cell line 293 and stable cell lines derivedtherefrom (GFP-LC3-HEK and p62-RL-HEK), as well as neuroblastoma cellline SH-SY5Y were cultured in minimum essential medium supplemented with10% (v/v) heat-inactivated fetal bovine serum, 50% (v/v) F-12 nutrientmixture, and 1% (v/v) antibiotic mixture comprised of penicillin andstreptomycin. Cells were kept at 37° C. in a humidified atmosphere of 5%CO₂. SH-SY5Y cells were plated at a density of 1×10⁵ viable cells perwell in 96-well plates for future analysis.

Animals

C57BL16 male mice were purchased from the National Laboratorial AnimalCenter (Taipei, Taiwan, R.O.C.) and kept at the Experimental AnimalFacility of the Institute of Cellular and Organismic Biology (ICOB) atAcademia Sinica, in which each mice was housed in individuallyventilated cage (IVC) with a 12:12 hr light-dark cycle with food andwater provided ad libitum. Experimental procedures for the describedanimal study were approved by the Institutional Animal Care andUtilization Committee (IACUC) of Academia Sinica (Taipei, Taiwan,R.O.C.). Mice were.

Assay on Tauopathy Toxicity

The ability of wild-type human Tau protein to induce cell death indeveloping neuronal cells was investigated using GFP-hTau fusion proteinunder the control of neuron-specific HuC promoter (Park et al., 2000).The expression construct was injected into zebrafish embryos at 1-cellstage. GFP-labeled neuronal cells were observed at 24, 48 and 72 hpf byuse of a fluorescence microscope. Some GFP signals were observed inneuronal cells of 24 hpf embryos, then broken down as small dots anddiminished in 48 hpf embryos. However, there were other GFP signalsremained intact in neuronal cells. Neuronal numbers with GFP signals inembryos at 72 hpf were counted and separated into two groups, lower than2 neurons (0-2) and more neurons (between 3-5).

Assay on Neurite Outgrowth Activity

To test whether BVEE can promote neurite outgrowth in zebrafish embryos,the expression plasmid pHuC-GFP, in which the GFP protein was expressedunder the control of a neuron-specific HuC promoter, was first injectedinto zebrafish embryos at one-cell stage. Eight hours after injection,those injected embryos were treated with BVEE or DMSO as control.Another 40 hr after treatment, zebrafish numbers with significantneurite outgrowth were counted.

Cell Viability Assay

HEK or derived stable cell lines (GFP-LC3-HEK and p62-RL-HEK) (5×10⁴cells/100 μL/well) were seeded onto each wells of 96-well microplates inculture medium containing respective compounds as specified andincubated at 37° C. for 24 hr. Viable cells were determined using theCellTiter 96 AQueous non-radioactive cell proliferation assay (Promega)in accordance with protocols described in the manufacturer's manual.Briefly, after the addition of the combined3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazine methosulfate solution (20 μL/well),microplates were incubated for 3 hr at 37° C. The conversion of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt into formazan in viable cells was quantitated by theabsorbance at 490 nm using a fluorescience/luminescence microplatereader (SpectraMax M5, Molecular Devices). The number of viable cells inculture was directly proportional to the absorbance at 490 nm. Viablecells in culture medium containing vehicle alone (0.1% DMSO, control)were referred to as 100% viability.

Cell-Based Reporter Assay for the Screening of Autophagy-ModulatingAgents

To generate a cell-based p62 degradation assay specific for autophagicactivity, stable clone of HEK cells (p62-RL-HEK) that constitutivelyexpress p62 C-terminally fused with Renilla luciferase reporter (p62-RL)was generated. The Renilla luciferase signal in p62-RL-HEK cells couldthus be measured to determine the steady level of p62-RL in the cells.Our studies confirmed that the increase of luciferase signal wasresulted from the inhibition of autophagy-mediated degradation of p62,while the decrease of luciferase was due to the induction ofautophagy-mediated p62 degradation. The efficiency of this cell-basedassay was evaluated by an autophagy inhibitor 3-methyladenine (3-MA) andan autophagy inducer rapamycin, respectively.

To identify effective herbal extracts that contain active ingredients ofautophagy modulators, p62-RL-HEK cells (2×10⁵ cells/well) were seededonto 96-well microplates and incubated at 37° C. for 2 days, followed bytreatments with 200 μg/mL of individual herbal extract for 24 hr at 37°C. Luciferase signals were determined by the addition of coelenterazine,the cell-permeable substrate of Renilla luciferase, to a finalconcentration of 5 μM, and were then quantitated by a VictorLightluminescence/fluorescence plate reader (Perkin Elmer). The luciferasesignal emitted by p62-RL-HEK treated with vehicle alone (0.1% DMSO) wasreferred to as 100% Relative Luc. Cells that are treated with either3-MA (5 μM) or rapamycin (200 nM) were included as positive controls.

Our data demonstrated that p62-RL-HEK cells treated with 3-MA exhibiteda significant increase of luciferase signal, which was an indicative ofaccumulation of p62 due to inhibition of autophagy by 3-MA. On the otherhand, p62-RL-HEK cells treated with rapamycin exhibited a significantdecrease in luciferase signal, consistent with the accelerateddegradation of p62 resulted from rapamycin-elicited activation ofautophagy. Herbal extract that induced greater alterations in RelativeLuc as compared to that by 3-MA or rapamycin was identified as potentialautophagy modulator.

Autophagic Activity Assay Using a GFP-LC3-Expressing Cell Line

HEK293 cells stably transfected with a GFP-tagged LC3 expressionconstruct was generated and verified for the constitutive expression ofthe recombinant GFP-LC3. A single clone derived from a homogenouspopulation of GFP-LC3-expressing HEK293 cells was isolated and named asGFP-LC3-HEK. To examine autophagy activity, GFP-LC3-HEK cells wereseeded onto 6-well microplates and treated with various concentrationsof BVEE for 24 hr. Respective treatments with 3-MA (5 μM) and rapamycin(200 nM) were included as positive controls. Clarified lysates derivedfrom BVEE-treated cells were analyzed by SDS-PAGE and western blot usinganti-p62, anti-LC3, and anti-GAPDH antibodies.

Neprilysin Activity Assay

SH-SY5Y cells were grown to confluence in 100 mm culture dishes, thenwere seeded in 12-well plates (1 mL; cell density 1×10⁶ cells/mL). Afterplating for 1 day, the medium was replaced with fresh DMEM/F-12supplemented with 10% FBS with or without the indicated compound(concentration from 0.25 to 30 μg/mL), then the cells were incubated foranother 24 hr, after which the medium was replaced with 200 μL assaybuffer (PBS containing 5.5 mM D-glucose, 0.3 mM sodium pyruvate, 25 mMsodium bicarbonate, and 1.5 μM zinc sulfate) containing 4 μM qf-Aβ(1-7)Cand the cells were incubated for 1.5 hr. To measure Aβ-degradingactivity, 150 μL of the assay buffer was taken for fluorescencemeasurement on a SpectraMax Gemini EM (Molecular Devices, USA) withexcitation at 346 nm and emission at 442 nm.

Intracerebral Aβ42-Injection Mouse Model of AD

To generate the acute Aβ42-induced mouse model of AD, 8-wk old C57BL/6male mice were intraperitoneally anesthetized with 40 mg/kg sodiumpentobarbital, followed by the injection of aggregated Aβ42 into thedorsal hippocampus using a 26-gauge needle connected to a microsyringe(Hamilton). The coordinates of the stereotaxic surgery were as follows:2 mm posterior to the bregma, 2.1 mm bilateral to the midline, and 1.8mm ventral to the skull surface. The volume of injection was 1 μL ofaggregated Aβ42 or 1 μL PBS. Mice subject to surgical procedures wereallowed to recovery for 7 days. The Aβ42 aggregate was prepared from asolution of 10 mM of soluble Aβ42 in 0.01 M PBS, pH 7.4. The solutionwas incubated at 37° C. for 3 days to form the aggregated Aβ42 andstored at −70° C.

Cognitive Function Measurements of a Injection Mouse Model

The cognitive function of Aβ42-injection AD mice was assessed by Morriswater maze. Briefly, Aβ42-injection AD mice were orally administeredwith either vehicle alone (0.1% DMSO) or 250 mg/kg BVEE daily for 2months. C57BL/6 male mice receiving sham operation or intracerebrallyinjection of PBS were fed with vehicle alone and served as untreatedcontrols. At the conclusion of dosing regime, mice were put into thewater maze from 4 different quadrants, and the spatial memory of eachmice was independently assessed by the average latency time each animaltook to find the hidden platform. This cognitive functional test wasconsistently conducted during the last 4 hours of the day portion of thelight cycle within a confined environment exposed to minimaldisturbance. The spatial acquisition trial was conducted daily for 5consecutive days.

Immunohistochemical (IHC) Analysis

To validate the anti-AD effects of BVEE that would not elicitsignificant neurotoxicity, the phenotypical changes of autophagyactivation in brain of treated and control animals were determined byimmunohistochemistry. Prior to being sacrificed, animals were perfusedwith 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brainswere cryosectioned horizontally into 15 μm sections that were mounted ongelatin-coated slides, followed by incubation with ablocking-permeabilizing solution (5% donkey serum and 0.25% TritonX-100) for 1-3 h. BVEE-induced autophagy activation in animals werevisualized by anti-p62 antibodies. Fluorescent signal was acquired by alaser confocal microscope (Leica TCS-SP5-MP).

Animal Experiment Using AD Double Transgenic Mice

All experiments protocols were approved by the Institutional Animal Careand Use Committee (IACUC) of Academia Sinica. APP/PS1 transgenic mice(B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/Mmjax) and non-transgenic littermatecontrols were used in the following experiments. To stabilize thegenetic background, the APP/PS1 mice were backcrossed with the wild-type(WT) C57BL/6J mice in the National Laboratory Animal Center. Genomic DNAof the APP/PS1 mice and the WT littermates were isolated by ZR GenomicDNA™ Tissue MiniPrep kit (Zymo Research Corp.). Human APPswe andPSENIdE9 genes were then amplified by polymerase chain reaction (PCR).Primers for APPswe were 5′-AGGACTGACCACTCGACCAG-3′ (SEQ ID NO: 1) and5′-CGGGGGTCTAGTTCTGCAT-3′ (SEQ ID NO: 2) while those for PSEN1dE9 were5′-AATAGAGAACGGCAGGAGCA-3′ (SEQ ID NO: 3) and 5′-GCCATGAGGCACTAATCAT-3′(SEQ ID NO: 4). Amplified DNA fragments were then visualized by 1.5%agarose gel electrophoresis with SYBR® Safe DNA Gel Stain (Invitrogen).Lyophilized BVEE powder (provided by Industrial Technology ResearchInstitute, ITRI) was dissolved in N-Methyl-2-pyrrolidone (NMP) as the10× BVEE stock (200 mg/mL), which was prepared twice a week and storedat 4° C. before dilution. The 10×BVEE stock were then diluted 10-foldswith the pre-mixed kolliphor/saline solution (1: 3.5, v/v) before thegavage administration. The APP/PS1 mice received 250 mg/kg/day of BVEEor saline treatment by gavage administration six days/week. Dosage wasadjusted once a month according to the body weight, which was recordedonce a month.

Evaluation of Long-Term BVEE-Treated APP/PS1 Mice on Cognitive Abilityby Monis Water Maze

Mice were subjected to the Morris water maze test at the age of 11months. The apparatus of the Morris water maze consists threecomponents: one water tank (100 cm diameter and 35 cm height), onetransparent circular platform (10 cm diameter), and the TopScanLitetracking system (CleverSys Inc). One black triangle, red square, bluecircle, and green star were respectively placed at the north, west, eastand south side of the tank wall as visual cues. The tank was filled withwater and milk, and therefore the transparent platform hidden 2 cmbeneath the water surface was invisible. The submerged platform wasplaced in the quadrant 3 of the tank and kept in the same locationduring the spatial acquisition trial. The Morris water maze test wasconsisted of two trial phases: the spatial acquisition trial (Day 1 toDay 5, four trials per day) and the probe trial on Day 6.

Start positions for individual trial were selected semi-randomly asdescribed in a previous study (Vorhees C V and Williams M T (2006) NatProtoc 1(2):848-858). The mouse was gently placed in the start positionand allowed to swim for 90 sec to find the hidden platform. The latencytime was recorded if the mouse had reached the platform within 90 secand stayed on the platform for 30 sec. Mouse failing to find theplatform within 90 sec time limit was guided to the platform by puttingan orange flag on the platform for an extra 30 sec. The mice who foundthe platform in this time period were allowed to stay on the platformfor another 30 sec, while the mice who failed to find the platform weredirectly put on the platform for 30 sec. After finishing the trial,these mice could return to their home cage and then rest for at least 15min until beginning of the next trial. Hair dryer and the tissue paperwere used to keep the mouse warm and avoid hypothermia during the wholecognitive test. Twenty-four hours after the last day of the spatialacquisition trial, the platform was removed from the water tank for aone-day probe trial. Each mouse received a 60-sec trial and the spatialmemory was evaluated by recording the latency time of the location ofhidden platform.

ELISA

After the Morris water maze test, five to seven mice from each groupwere sacrificed and their hippocampus, cerebral cortex were collected,aliquoted and stored at −80° C. for subsequent ELISA assay. Collectedbrain samples were homogenized in 400 μL TBS buffer (50 mM Tris, 2 mMEDTA, 150 mM NaCl, protease inhibitor cocktail (Sigma P8340), pH 7.4)with 1 mL Duall tissue grinder with PTFE pestle and centrifuged at 4° C.for 20 min at 15000 rpm (20000×g). The insoluble pellet fractions werecollected and then resuspended in 400 μL 70% formic acid, sonicated for1 min, and centrifuged at 4° C. at 15,000 rpm (20000×g) for another 20min. Supernatant were equilibrated (1:20) with neutralization buffer (1M Tris, 500 mM Na₂HPO₄), aliquoted and collected as the “insoluble Aβfraction”.

Insoluble human Aβ40 and Aβ42 levels of the collected hippocampus andcerebral cortex were quantified by commercial human Aβ40 (KHB3482,Invitrogen) and Aβ42 (KHB3442, Invitrogen) ELISA Kit. Aβ40 and Aβ42detections were conducted according to manufacturer's instructions.Total protein concentration of the insoluble Aβ fractions was quantifiedby the Bradford method. The insoluble Aβ fractions were then diluted atthe ratio of 1:500 and 1:1,000 for Aβ40 and Aβ42 detection,respectively. Diluted insoluble Aβ fraction and primary detectionantibody were added into wells and incubated at room temperature for 3hours with shaking. After washing, horseradish peroxidase(HRP)-conjugated secondary antibody solution was supplied at roomtemperature for 30 min with gentle agitation. Stabilized chromogensolution for Aβ40 and Aβ42 detection was added at room temperature for30 min and 15 min, respectively. Reactions were terminated by the stopsolution, and the absorbance at 405 nm was measured immediately.

ThS Staining

After the Morris water maze test, three mice of each group wereeuthanized by cervical dislocation. Three mice were perfused with PBS(136.89 mM NaCl, 2.68 mM KCl, 1.62 mM KH₂PO₄, and 10.14 mM Na₂HPO₄, pH7.4) buffer and 4% paraformaldehyde (PFA)/PBS. Perfused mice brain werethen post-fixed in 4% PFA/PBS with gentle shaking at room temperaturefor another 24 hours. After post-fixation, brain samples were wash withPBS buffer and preserved in 70% ethanol/water solution until theparaffin embedding protocol.

PFA-fixed brain tissues were dehydrated by a semi-enclosed benchtoptissue processor (Leica TP1020). Paraffin blocks containing thedehydrated brain samples were prepared by Leica EG1150 H. Coronalsections with 3 μm thickness were cut on a rotary microtome (LeicaRM2235). Two brain sections were picked up with a paint brush, put ontothe surface of a water bath, floated onto the surface of clean glassslides, and placed on a 34° C. warming block for several hours.

Paraffin slides were de-paraffinized and rehydrated with xylene,absolute ethanol, 95% ethanol, 70% ethanol and water, sequentially.Rehydrated slides were applied with 1% (w/v) thioflavin S solution for10 min at room temperature protected from light. Wash the slides with80% ethanol and water to remove excess fluorochrome and facilitatevisualization. Thioflavin S-positive signals were then visualized in afluorescence microscope, equipped for evaluation of green fluorescenceand the plaque number, plaque area and plaque size were analyzed byImageJ.

Liver and Kidney Toxicity Test

Blood samples were collected through the orbital sinus beforeeuthanasia. Collected blood samples were incubated on ice for at leastan hour, and centrifuged at 1500×g for 10 min (4° C.). Supematant serumsamples were collected, aliquoted and frozen in −30° C. Serum aspartatetransaminase (AST), alanine transaminase (ALT), alkaline phosphatase(ALP), blood urea nitrogen (BUN) and creatinine (CRE) were then analyzedby Fuji Dri-chem 4000i Analyzer (Taiwan Mouse Clinic).

Example 1 Bauhinia Extract Decreases Tauopathy Induced Toxicity

In this example, the effect of the ethanol extract of Bauhinia variegate(BVEE), which was prepared in accordance with the procedures set forthin “Materials and Methods” section, on tauopathy toxicity was evaluatedby monitoring the expression of GFP-hTau fusion protein in zebrafishembryo. The zebrafish embryos at 1-cell stage was injected with theexpression construct pHuc-hTau-GFP, the expression of the Tau proteinwas then monitored by the co-expressed GFP signal. Using fluorescencemicroscope, GFP-labeled neuronal cells were observed at 24, 48 and 72hpf. Neuronal numbers with GFP signals in embryos at 72 hpf were countedand separated into two groups: lower than 2 neurons (0-2), and moreneurons between 3-5. Results are illustrated in FIG. 1.

Without BVEE treatment, 77% of embryos had 0-2 neurons and only 23%embryos with more neurons between 3-5. Conversely, BVEE treatmentdecreased tauopathy induced toxicity by increasing neuron expression inzebrafish embryos to 43% (FIG. 1, panel B).

Example 2 Bauhinia Extract Promotes Neurite Outgrowth

Similar to example 1, zebrafish embryo at 1-cell stage was injected withthe expression plasmid pHuC-hTau-GFP, then treated with BVEE or DMSO(the control) to see if BVEE may promote neurite outgrowth by countingthe number of sprouting neurons. Results are illustrated in FIG. 2.

As the data indicated, HuC promoter-driven GFP was expressed intrigeminal ganglia, axons, and interneurons of zebrafish embryos at 48hpf (FIG. 2, panel A). The percentage of injected zebrafish embryostreated with DMSO with neurite outgrowth was 25% (FIG. 2, panel B).Conversely, BVEE treatment induced significant neurite outgrowth, inwhich the percentage of zebrafish embryo with neurite outgrowth reacheda significant high level of 57% (FIG. 2, panel B).

Example 3 Effect of BVEE on the Activation of Autophagy

In this example, the ability of BVEE to induce autophagy was examined bymonitoring the conversion of LC3-I to LC3-II and the expression level ofp62 in cells treated with or without BVEE. Results are summarized inFIG. 3.

FIG. 3, panel A is a western blot analysis of the expression of LC3-I,LC3-II and p62. The data indicated that BVEE-treated cells exhibitedsignificant increases in the level of LC3-II, concomitant with asignificant decrease in that of p62. Consistently, BVEE treatments alsoresulted in a dramatic reduction in p62 in a cell-based luciferasereporter assay of autophagy activity (FIG. 3, panel B). These datastrongly suggest that BVEE of the present disclosure contains activeingredients that activate autophagy.

Example 4 Effect of BVEE on Neprilysin (NEP) Activity

In this example, the effect of BVEE on NEP activity was examined bymonitoring the level of a peptide substrate, qf-Aβ(1-7)C peptide, inwhich a fluorophore and a quencher are respectively linked to itsC-terminus and N-terminus. The existence of the quencher quenches thefluorescence. When qf-Aβ(1-7)C is cleaved by NEP, the quencher and thefluorophore are separated and strong fluorescence is emitted from thefluorophore. NEP is a type II membrane protein, and if the membrane ofthe cells (e.g., SH-SY5Y or N2a cells) contained higher amounts of NEP,then higher amounts of qf-Aβ(1-7)C peptides would be digested andthereby resulting higher fluorescence intensity. Results are illustratedin FIG. 4.

As evident from FIG. 4, there is a clear dose-dependency between the NEPactivity and BVEE concentration, for NEP activity increases as theconcentration of BVEE increase, suggesting that BVEE treatment maypromote NEP-dependent clearance of Aβ.

Example 5 BVEE does not Elicit any Significant Cytotoxicity

In this example, the toxicity of BVEE preparation of the presentdisclosure was determined by use of a cell viability assay. Results areprovided in FIG. 5.

As depicted in FIG. 5, cell viability remained relatively comparable tothat of the control in cells treated with either low (50 μg/mL) or highconcentration (100 μg/mL) of BVEE. The result clearly indicated thatBVEE of the present disclosure does not possess any toxicity towardp62-RL-HEK cells, thus is safe to use as a dietary supplement.

Example 6 BVEE Treatment Induced Cognitive Improvement in anIntracerebral Aβ42-Injection Mouse Model of AD

In this example, the efficacy of BVEE of the present disclosure oncognitive improvement was evaluated in an acute Aβ42-induced AD mousemodel in accordance with procedures described in “Materials and Methods”section. Results are summarized in FIGS. 6 and 7.

As evident from FIG. 6, significant cognitive improvement was found inAβ-injected mice treated with BVEE, whereas those treated with vehiclealone displayed a marked cognitive deficit as compared to sham-operatedanimals. Consistently, a dramatic increase in the induction of autophagywas found in the hippocampus of mice treated with BVEE, which wasevidenced by a significant decrease in the expressed level of p62 (FIG.7).

Taken together, the findings support the proposition that BVEE of thepresent disclosure contains active ingredients that activate autophagy,which in term suppresses Aβ42-elicited neurotoxicity and improvesspatial learning and memory of the test animals.

Example 7 Effect of BVEE on AD Double Transgenic Mice

In this example, effect of BVEE on the spatial and reference memory ofthe test animals (i.e., APP/PS1 mice) were evaluated by the Morris watermaze test in accordance with the procedures described in “Materials andMethods” section. Results are summarized in FIG. 8.

On the first day (Day 1, FIG. 8), the APP/PS1 mice received both salineor BVEE treatment exhibited similar escape latencies in reaching theplatform as the WT littermate, which indicated that these mice hadsimilar learning ability and no quadrant preferences after the BVEEtreatment. Conversely, the saline-treated APP/PS1 mice demonstratedimpaired performances with longer escape latencies to locate the hiddenplatform. 75% of the mice in this group (9 out of 12 mice) were unableto locate the hidden platform on the first day of the test. After fivedays of successive trainings, some of the APP/PS1 mice were able toreach the platform within the 90 sec time limit, yet their escapelatencies remained at about 60 sec. On the other hand, the WT mice,which did not develop AD symptoms during their lifespan, quickly learnedto locate the platform after the second trial on Day 1 (12 out of 16mice). Significant decline of escape latencies between the WT andAPP/PS1 mice were observed after Day 2. Furthermore, 94% of the WT mice(15 out of 16 mice) were able to locate the platform within 33 sec afterfinishing the spatial acquisition trials. Compared with the salinetreatment group, mice receiving BVEE treatment exhibited remarkablecognitive improvement after five days of successive training (P<0.01),their escape latencies (i.e., BVEE-treated APP/PS1 mice) on Days 2 and 3decreased significantly by 50%. Although the latency increased on Day 4,yet it was still lower than that of the saline-treated APP/PS1 mice. Onday 5, the escape latency of the BVEE-treated mice were similar to thatof the WT mice, and was significantly shorter than that of thesaline-treated APP/PS1 mice. After finishing the 5-day spatialacquisition trial, mice were subjected to a one-day probe trial, whichwas used to evaluate whether they had learned to locate the platform byrecording the escape latencies, results are depicted in FIG. 8, panel B.Similar to the results found in the spatial acquisition trial, thesaline-treated mice exhibited impaired cognitive capability in the probetrial (about 50 sec). Conversely, the BVEE-treated mice exhibitedsimilar cognitive ability as that of the WT littermate.

After finishing the cognitive test, the APP/PS1, BVEE and WT mice wereeuthanized at 11 months of age, and the insoluble human Aβ40 and Aβ42levels of the hippocampus and cerebral cortex were quantified by ELISA.Results are illustrated in FIG. 9, panel A. Abundant Aβ40 and Aβ42plaques were found in both hippocampus and cerebral cortex of thesaline-treated APP/PS1 mice, as compared to that of the WT mice. Minordecline of the β-amyloid deposits in hippocampus were observed (25%decrease in Aβ40 and 21% decrease in Aβ42) after the BVEE-treatment,which had been administrated for 8 months. On the other hand, theinsoluble Aβ40 and Aβ42 deposits in the cerebral cortex were remarkablyreduced by 46% and 33%, respectively (P<0.01). Consistent to thisfinding, marked reduction in amyloid plaques quantified by ThS stainingwas also found in cortex, and significant decrease in plaque number wasobserved in hippocampus (FIG. 9, panels B and C).

Example 8 Effect of BVEE Treatment on the Function of Liver or Kidney

In this example, long term effect (i.e., 8-month) of BVEE on the testanimals were evaluated by monitoring their liver and kidney functions.Liver function was determined by measuring the serum levels of variousmetabolic enzymes, which include aspartate transaminase (AST), alaninetransaminase (ALT) and alkaline phosphatase (ALP); whereas the renalfunction was determined by measuring the serum levels of blood ureanitrogen (BUN) and creatinine (CRE). Results are depicted in FIG. 10.

The data in FIG. 10 indicated that BVEE treatment did not significantlyaffect any of the ALT, BUN and CRE levels. However, a significantlyhigher level of AST and a significantly lower level of ALP were found inBVEE treated animals, as compared with those of the saline-treatedcontrol. Yet, none of the BVEE-treated animals exhibited pathologicalsymptoms. Thus, we concluded that long-term BVEE oral administration forat least 8-months would not affect the liver and/or kidney functions ofthe test animals.

Example 9 Comparison of the Efficacy of Present Extracts on NEP Activityand Autophagy Induction

In this example, the efficacy of the present extracts ofBauhinia×blakeana Dunn. on NEP activity was examined. The extracts wereprepared in accordance with the procedures described above in the“Materials and Methods” section, in which 95% ethanol, 40% ethanol orhot water was used as the solvent to extract stem powders ofBauhinia×blakeana Dunn. Results are illustrated in FIG. 11.

As evidenced in FIG. 11, the extracts of Bauhinia×blakeana Dunn.prepared by extraction with 95% ethanol (BBEE), 40% ethanol (BB40EE), orhot water (BBWE) exhibited increased NEP activity than that of thecontrol.

Example 10 Effects of the Present Extracts Obtained by Various Types ofSolvents on the Induction of Autophagy

In this example, the efficacy of the present extracts of B. variegate onthe induction of autophagy in p62-RL-HEK cells were examined, in whichthe extracts were respectively obtained by use of extracting solventsthat include hexane, ether, ethyl acetate (EA), hot water and 95%ethanol. The present extracts of B. variegate were prepared inaccordance with the procedures described above in the “Materials andMethods” section. Results are summarized in FIG. 12.

The data indicated that, while all the solvents could successfullyextract the autophagy-inducing components from B. variegate, preferably,the autophagy-inducing constituents was isolated by use of organicsolvents, such as hexane, ether, ethyl acetate, or ethanol (FIG. 12).These results suggest that the active ingredients of B. variegate can beefficiently isolated in an ethanol-based to extraction procedure, whichmight be conducive to the formulation of BVEE as a dietary supplement.

It will be understood that the above description of embodiments is givenby way of example only and that various modifications may be made bythose with ordinary skill in the art. The above specification, examplesand data provide a complete description of the structure and use ofexemplary embodiments of the invention. Although various embodiments ofthe invention have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those with ordinary skill in the art could make numerous alterations tothe disclosed embodiments without departing from the spirit or scope ofthis invention.

What is claimed is:
 1. A method for manufacturing a Bauhinia spp.extract, comprising the steps of: extracting a powdered stem of Bauhiniaspp. with water or ethanol at a ratio from 1:5 (w/v) to 1:20 (w/v) atabout 10-100° C. for about 0.5 hours to 10 days to produce the Bauhiniaspp. extract.
 2. The method of claim 1, wherein the powdered stem ofBauhinia spp. is extracted with water at the ratio of 1:10 (w/v) atabout 50° C. for about 2 hours.
 3. The method of claim 1, wherein thepowdered stem of Bauhinia spp. is extracted with 95% ethanol (vol %) atabout 15-35° C. for about 1-10 days.
 4. The method of claim 3, whereinthe powdered stem of Bauhinia spp. is mixed with 95% ethanol (vol %) atthe ratio of 1:10 (w/v) at about 25° C. for about 7 days.
 5. The methodof claim 1, wherein the Bauhinia spp. is Bauhinia variegate orBauhinia×blakeana Dunn.